Accurate and rapid detection of microorganisms, such as bacteria and viruses, has important applications in many areas including biodefense, food safety, diagnostics, pathology, forensics and drug discovery. Many food products, for example, especially processed meat, vegetables and dairy products are probable carriers of potent food-borne pathogens, including E. coli, Salmonella, Listeria and Campylobacter jejuni. In fact, there have been numerous incidents of costly food product recalls across United States in past years, which might have been preventable or minimized if reliable early detection could have been made. As another example, in hospitals and other healthcare facilities, presence of pathogens can pose health risks to both patients and care takers. Thus, the microbial environment in these facilities must be constantly monitored to prevent disease transmission. The accurate diagnosis, prognosis and effective treatment of infectious diseases also rely on the precise identification of the pathogenic species responsible.
The classical method for microorganism detection is based on cell culture, where a sample is collected and then cultured to grow enough of the microorganism species to be detected. In the case of bacteria, colonies of the bacteria can be counted visually under a microscope. A major drawback of the culture method is that it can sometimes take days for the culture to grow. Another problem is that not all microorganisms can be cultured because some microorganisms can only survive under certain narrow conditions as may be defined by pH, temperature, nutrient composition and the co-presence of other microorganisms, for example. Immunology-based microorganism detection is one alternative method. In this method, proteins, such as toxins, associated with the microorganisms are detected using antibodies. However, this method suffers a general lack of high quality antibodies, and higher cost can be a problem. Another more recently developed method is based on the detection of microbial DNA or RNA using polymerase chain reaction (PCR). In this method, a nucleic acid sequence unique to a particular microorganism species is selectively amplified and analyzed. The PCR-based detection method is highly accurate and relatively fast, taking only a few hours, instead of days, to complete. The methods also permit simultaneous detection of multiple microorganism species when different fluorescently labeled probes are used. Despite some of the obvious advantages of these genetically based test methods, nucleic acid amplification is a required step, which has the problem of not being able to distinguish between nucleic acid from live cells and that from dead ones.
Recently, so-called viability PCR has been developed to overcome the live cell selectivity problem. A key to the technique is the use of a DNA modifier dye, propidium monoazide (PMA). PMA is a doubly-positively charged DNA binding dye with a photoreactive azido group, which upon photolysis undergoes crosslinking with DNA, thereby covalently modifying the nucleic acid with the dye. When the DNA is sufficiently modified, it loses its biological activity, being unable to serve as template in polymerase chain reaction. Another property of PMA is that in a typical viability PCR-based detection, a sample comprising both live and dead cells is first treated with PMA in the presence of light. Since dead cells have a compromised cell membrane, their DNA is exposed for PMA modification; whereas, viable cells, which have cell membrane to prevent PMA from getting into contact with their nucleic acid, are often unaffected by the treatment. In the subsequent step, viable cells are then lysed to expose their DNA for selective amplification.
Although PMA has proven useful for the detection of a number of microorganism species under some conditions, it does not work well or completely fails to work for some organisms under certain conditions. For example, some results indicate that relatively short amplicons are not affected by PMA treatment. Other results indicate that for Staphylococcus aureus samples collected by swabbing from surfaces, PMA does not allow the determination of amount of living cells, which has been attributed to PMA dye getting into viable cells that were collected using that particular procedure. It has also been reported that, when PMA qPCR was used to detect Mycobacterium avium subspecies paratuberculosis (MAP), a gram-positive bacterium, the dye was found to enter viable MAP, causing underestimation of the number of viable cells. PMA has also been found to enter viable Salmonella serovar Enteritidis and in the meantime ineffective in suppressing qPCR signal from the killed bacteria.